This invention relates to the field of plant genetic engineering and, more particularly, to the regeneration of complete plants containing full length copies of genetically engineered T-DNA and the transmission of T-DNA to R1 progeny.
Ti plasmids of the plant pathogen Agrobacterium tumefaciens have the unique natural ability to transform cells of susceptible host plants by the insertion of an 8 to 23 kilobase (kb) sector of plasmid DNA into host chromosomal DNA (Chilton et al., Cell 11:263-71 (1977); Chilton et al., Proc. Natl. Acad. Sci. USA, 77:2693-97 (1980); Willmitzer et al., Nature (Lond.) 287:359-61 (1980); Yadav et al., Nature (Lond.) 287:458-61 (1980); and Zambryski et al., Science 209:1385-91 (1980)). This transferred DNA (T-DNA) causes the transformed cells to synthesize new metabolites called opines (Petit et al., Physiol. Veg., 8:205-13 (1970);Bomhoff et al., Mol. Gen. Genet. 145:177-181 (1976); and Montoya et al., J. Bacteriol., 129:101-07 (1977)). The synthase enzyme for one opine, octopine, has recently been shown to be encoded by a T-DNA gene (Murai and Kemp, Proc. Natl. Acad. Sci. USA, 79:86-91 (1982); and Schroder et al., FEBS Lett., 129:166-68 (1981)). Additional genes in T-DNA affect the phytohormone balance of transformed cells as evidenced by altered morphology of tumor cells transformed by various mutant T-DNAs (Ooms et al., Gene, 14:33-50 (1981); and Garfinkel et al., Cell, 27:143-153 (1981)). Transformed plant cells containing wild type T-DNA grow in vitro without an exogenous supply of either auxin or cytokinin (Braun, Am. J. Bot., 34:234-240 (1947)), whereas normal plant cells usually require both substances for growth in culture. Mutations in one T-DNA locus cause tumors from which abundant roots proliferate ("rooty" mutants), while mutations in a second T-DNA locus cause tumors from which shoots proliferate ("shooty" mutants)(Ooms et al., supra, Garfinkel et al., supra). Transformation of tobacco cells by shooty mutant T-DNA results in tumors which exhibit a complex auxin requirement. Such tissues will grow on hormone free medium under conditions which allow shoot proliferation, but otherwise require auxin for continuous growth (Binns et al., Cell (1982)). The rooty and shooty functions map in a T-DNA region common to octopine, nopaline, "unusual nopaline" and agropine Ti plasmids of wide host range (Chilton et al., Nature, 275:147-149 (1978); Depicker et al., Nature, 275:150-52 (1978); Engler et al., J. Mol. Biol., 152:183-208 (1981); and Guyon et al., Proc. Natl. Acad. Sci., 77:2693-97 (1980), evidence that all such Ti plasmids promote oncogenic growth of plant cells through a common mechanism.
T-DNA acts as a natural gene vector for A. tumefaciens, producing transformed plant cells that display an abnormal hormonal balance and synthesize new metabolites. Recent work has shown that the Ti plasmid can be exploited as an artificial gene vector to introduce novel genes into plant tumor cells (Hernalsteens et al., Nature, 287:654-656 (1980); and Leemans et al., EMBO Journal, 1:147-52 (1982)). Heretofore, a major obstacle to the exploitation of Ti plasmids as gene vectors for higher plants has been the difficulty of regeneration of whole plants from transformed plant cell lines. Cloned teratomatous lines of tobacco cells containing wild type nopaline plasmid pTiT37 T-DNA spontaneously or upon cytokinin induction regenerated shoots that displayed varying degrees of normalcy upon grafting onto healthy host plants (Braun and Wood, Proc. Natl. Acad. Sci., 73:496-500 (1976); Turgeon et al., Proc. Natl. Acad. Sci., 73:3562-64 (1976); and Binns et al., Differentiation 19:97-102 (1981)). Such shoots synthesized nopaline (Wood et al., Differentiation 11:175-80 (1978)), failed to form roots, and were resistant to superinfection by A. tumefaciens (Braun and Wood, supra; Turgeon et. al., supra; and Binns et al., supra). When fertile, these grafted shoots produced seed that gave rise to apparently normal plants that lacked nopaline, produced roots and were sensitive to A. tumefaciens (Braun and Wood, supra; Turgeon et al., supra; Binns et al., supra). Indeed, the cells of one such plant were shown to be completely free from T-DNA (Yang et al., Mol. Gen. Genet., 177:704-14 (1980); Lemmers et al., J. Mol. Biol., 144:353076 (1980)). Similar results have been obtained in studies of tobacco cells transformed in vitro by either octopine or nopaline type T-DNA (Wullems et al., Proc. Natl. Acad. Sci. 78:4344-48(1981a); Cell, 24:719-28 (1981b)). In such experiments, opine-positive plant cells with roots were not obtained, and shoots obtained by grafting were usually both opine-positive and resistant to superinfection by A. tumefaciens. A single example has been reported of opine-positive complete plants regenerated from a crown gall tumor initially incited by a shooty mutant of octopine T-DNA (Leemans et al., supra). These plants segregated the octopine trait in Mendelian fashion to healthy progeny, evidence that T-DNA was situated in chromosomes of the parental tissue. However, the T-DNA in these plants was found not to be full length. A large deletion of the central part of T-DNA apparently gave rise to plant cells with little T-DNA except for the octopine synthase gene.
There has thus been a failure of past efforts to regenerate whole plants containing intact T-DNA. Such efforts have produced instead plants whose T-DNA has been almost completely deleted.